Comparison of radiometric macrophage assay and fluorescein diacetate/ethidium bromide staining for evaluation of M. leprae viability

Earlier studies from our laboratory reported that a radiometric Mycobacterium leprae resident macrophage assay was a useful in vitro indicator of bacillary viability with good correlation with the established mouse foot pad model. The present study compares our assay with the recently described fluorescein diacetate/ethidium bromide (FDA/EB) method. M. leprae extracted from the dermal lesions of 73 bacilliferous leprosy patients were tested concurrently by both techniques. Good correlation (r = 0.52, p less than 0.001) was found between the radiometric assay evaluating DNA synthesis and the FDA/EB staining reflecting the presence of active esterase enzyme. In addition, the utility of the FDA/EB staining in the monitoring of therapy was established. Twenty-two patients treated for greater than 1 year showed lower numbers of green fluorescing bacilli when compared to 19 untreated or short-term-treated individuals.     

Fluorescent method (fluorescein diacetate and ethidium bromide) to study the viability of Cryptococcus neoformans in liquor

The utilization of the fluorescent method (fluorescein diacetate DF and ethidium bromide BE), to verify the viability of fungal cells, was studied in 40 samples of liquor, from patients with neurocryptococcosis. For removing leukocytes and red blood cells, which produce interfering fluorescence, good results were obtained with 0.3% saponin solution. After processing of liquor, 0.1 ml aliquots of resulting suspension were mixed to equal volumes of fresh DF-BE solution. The best incubation period for staining was 30 minutes, resulting in good differentiation between viable (green fluorescence) and non viable (red fluorescence) cells.     

Effect of presensitization with BCG and Mycobacterium leprae on granuloma formation to M. leprae

Granulomas which develop in draining lymph nodes, following the intradermal injection of cobalt-irradiated Mycobacterium leprae into the ear of the guinea pig 2 and 5 weeks earlier, were studied in animals which had been presensitized with BCG vaccine or M. leprae and compared with granulomas that developed in previously unsensitized guinea pigs. Presensitization with mycobacteria accelerated the development of the granulomas. Granulomas in previously unsensitized guinea pigs were found ultrastructurally to contain phagocytosing macrophages similar to those in lepromatous leprosy, and M. leprae presensitization did not alter the type of granuloma found. Those in BCG-presensitized guinea pigs contained secretory epithelioid cells with rough endoplasmic reticulum similar to those found in borderline tuberculoid leprosy or reversal reactions. The significance of these findings in relation to the current use of vaccines in leprosy is discussed.     

Infection of the congenitally athymic rat with Mycobacterium leprae

The susceptibility of congenitally athymic rats to Mycobacterium leprae infection has been investigated. Following inoculation of small numbers of M. leprae (5 X 10(3] into the foot pad, the organisms replicated and attained a maximum of 2.6 X 10(8) per foot pad at 294 days; there was limited dissemination to the tail. In similarly inoculated neonatally thymectomized Lewis rats (NTLRs) a ceiling of 2 X 10(7) organisms was reached. When a larger inoculum (10(7] was given, the number of bacilli in athymic rat foot pads peaked at 6.7 X 10(8) and after approximately 240 days a plateau of between 2 X 10(8) and 6 X 10(8) per foot pad was reached. Dissemination to superficial tissues occurred approximately nine months after inoculation, when significant numbers of bacilli were recovered from the foot pads, ears, snout, and tail. Following intravenous inoculation of 10(7) M. leprae into athymic rats, significant numbers of bacilli were recovered from the superficial tissues by 300 days post inoculation. The numbers of organisms reached a plateau of about 10(8) by one year. Autopsy of infected animals from 1-2 years after inoculation revealed no gross abnormalities except for a purulent bronchitis and bronchopneumonia. Although normal grossly, the ears, tail, snout and foot pads showed a varying degree of infiltration by histiocytes. In some this was almost imperceptible, in others there were large accumulations of foamy macrophages reminiscent of lepromatous leprosy. The numbers of mycobacteria present in Fite stains ranged from 2+ (several organisms or clusters of organisms) to 5+ (very numerous). The lymph nodes contained numerous non-caseating granulomata composed of activated macrophages which contained large (4+) or very large (5+) numbers of bacilli. Mycobacteria were present in the cells of the mononuclear-phagocyte series in the liver and spleen of animals killed 12-15 months post inoculation, but were absent from these cells in animals killed later. M. leprae were also numerous in the smooth muscle of the scrotum. It is concluded that congenitally athymic rats are highly susceptible to M. leprae infection. Despite their lack of thymic-dependent T cell function, it appears that they possess the defense mechanism(s) capable of limiting the infection.     

Crossreactivity between Mycobacterium leprae and various actinomycetes and related organisms

Serological crossreactivity was analyzed between M. leprae and strains of various species of Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, and related organisms. M. leprae shares antigens with most of these organisms, and sera from patients with lepromatous leprosy contain antibodies against them. The results demonstrate that M. leprae shares more antigens with the mycobacteria than with strains of the other tested genera, thus supporting the view that the leprosy organism belongs to the genus Mycobacterium. One precipitinogen (designated p beta) was found to be common to M. leprae and the streptomycetes, and sera from patients with lepromatous leprosy contain antibodies against this antigen.     

Separation of Mycobacterium leprae from contamination with armadillo-liver-derived "pigment" particles

Mycobacterium leprae isolated from armadillo liver by the widely used IMMLEP protocol is sometimes contaminated with a particulate "pigment." This paper describes a simple, efficient, and rapid method for purifying large quantities of contaminated bacteria, which may readily be used as an additional step added at the end of the protocol when necessary. The process involves a discontinuous Percoll gradient and generates an essentially pure fraction containing greater than 90% of the original bacteria, and a fraction of "pigment" slightly contaminated with bacteria. Use of the system should release large additional numbers of pure M. leprae suitable for use in human vaccine trials.     

Ultrastructural features of macrophages of armadillos infected with actively multiplying Mycobacterium leprae

Experimental leprosy lesions in the armadillo (Dasypus novemcinctus) were studied by freeze etching and ultrathin sectioning. Infected macrophages have distinct intracytoplasmic foamy structures in the form of spherical droplets accumulated around multiplying bacilli. This finding is the same as those observed in human lepra cells and nude mice macrophages infected with M. leprae.     

Whole-genome expression analysis of Mycobacterium leprae and its clinical application

The whole-genome sequence analysis of Mycobacterium leprae, which was completed in 2001, revealed the characteristics of this microbe's genomic structure. Half of the M. leprae genome consists of a limited number of protein-coding genes and the rest comprises non-coding regions and pseudogenes. We performed membrane array and tiling array analyses to analyze the gene-expression profile of the M. leprae genome and found that pseudogenes and non-coding regions were expressed similarly to coding regions at the RNA level. The RNA expressions were confirmed by real-time PCR analysis. Expression of these RNAs in clinical samples showed varying patterns among patients, thus indicating that the analysis of RNA expression patterns, including non-coding regions and pseudogenes, may be useful for understanding the pathological state, prognosis, and assessment of therapeutic progress in leprosy.